Proteomic Examination for Gluconeogenesis Pathway-Shift during Polyhydroxyalkanoate Formation in Cupriavidus necator Grown on Glycerol.

Abstract

Because of availability and inexpensiveness, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by Cupriavidus necator was found to produce lower PHA with low bacterial growth rates. According to our study, C. necator was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the Cupriavidus necator H16 experiment give rise to the break down the bonds of α,α-1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (C6H14O6), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs.